Antimicrobial resistance profile of Staphylococcus aureus isolated from patients, healthcare workers, and the environment in a tertiary hospital in Addis Ababa, Ethiopia

Staphylococcus aureus infection and colonization in patients may be transmitted to healthcare providers and the environment and subsequently cause healthcare-associated infections in other patients. Pathogenic S. aureus strains produce virulence factors, such as Panton-Valentine Leukocidin (PVL), that contribute to the severity of infections and aid in their spread. The emergence of antimicrobial resistance (AMR) is additional concern with respect to S. aureus infection. In this study, the virulence genes and antibiotic resistance profiles of S. aureus were characterized from patients’ clinical isolates, healthcare workers’ (HCWs’) nasal colonization screenings, and the environment at a tertiary healthcare hospital in Addis Ababa, Ethiopia. A total of 365 samples were collected from September 2021 to September 2022: 73 patients’ clinical specimens, 202 colonization screenings from HCWs, and 90 hospital environment’s swabs. Fifty-one (25.2%) HCW and 10/90 (11.1%) environment S. aureus isolates were identified. Among the 134 isolates, 10 (7.5%) were methicillin-resistant S. aureus (MRSA). Three (4.1%), five (9.8%), and two (20.0%) of the MRSA isolates were identified from the patients, HCWs, and the environment, respectively. Overall, 118 (88.1%) were ampicillin and penicillin resistant; 70 (52.2%) were trimethoprim sulfamethoxazole resistant; and 28 (20.9%) were erythromycin resistant. S. aureus isolates from patients were more resistant to antibiotics than isolates from HCWs or the hospital environment (p<0.05). A total of 92/134 (68.6%) isolates possessed the lukfF-PV gene, which was identified in 62 (85.0%), 26 (51.0%), and 4 (40.0%) of the patient, HCWs, and the environment, respectively. The proportion of lukfF-PV gene containing S. aureus isolated from patient samples was statistically significant. Four (40.0%) of the MRSA isolates also had the lukfF-PV gene. The identification of highly AMR and virulence factors from patients, HCWs and the environment is concerning. Further studies are needed to identify potential transmission links and improve infection prevention and control.


Introduction
Staphylococcus aureus is a widespread organism that is also found as a normal flora of human body.Approximately 20-30% of the human populations are reported to be colonized with persistent nasal carriage of S aureus, while 60% are intermittent carriers, and another 20% are non-carriers [1,2].S. aureus colonization of patients has been connected to subsequent infection [3], and healthcare workers (HCWs) have been identified as potential reservoirs and possible source for the cross-transmission of pathogens associated with healthcare-associated infections (HAIs) [4,5].
S. aureus infections can affect any part of the body from minor skin infections to serious, life-threatening pneumonia and bacteremia [6,7].The production of virulence factors by pathogenic strains, such as expression of a cell-surface protein that binds and inactivates antibodies, as well as the production of powerful protein toxins like staphylococcal enterotoxin and Panton-Valentine Leukocidin (PVL), can aid in the spread of infections [8] PVL is a poreforming toxin largely responsible for skin and soft tissue infection.There is a significant variation in the prevalence of PVL among methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) infections worldwide.The prevalence of PVL toxin has been reported to be increasing in Africa [9,10].
Emergence of antimicrobial resistance (AMR) in healthcare facility is a growing concern due to increasing prevalence of multidrug resistant organisms (MDROs) and limited availability of effective antimicrobial therapeutics for treatment.In Ethiopia, the majority of studies on S. aureus AMR patterns rely on culture-based pathogen identification and drug susceptibility testing.However, there is a limited understanding of the molecular resistance and virulence gene profiles of S. aureus in Ethiopia [14].This study aimed to characterize the antibiotic resistance and virulence gene profiles of S. aureus isolates obtained from patient clinical samples, healthcare worker colonization screenings, and environmental sampling at a tertiary healthcare hospital in Addis Ababa, Ethiopia.

Study design, sampling procedures and data collection
This cross-sectional study was conducted at St Paul's Hospital Millennium Medical College (SPHMMC) in Addis Ababa, Ethiopia.Patients' clinical specimen, healthcare workers' nasal swab and hospital environment swabs were the source of samples utilized in this study.
Patients: All clinical specimens submitted for routine culture that was positive for S. aureus at the SPHMCC from September 2021 to September 2022 were included in the study.The isolates were kept at -80 freezer using 20% glycerol and tryptic soya broth in a cryo-tube for further testing.Patient demographics, diagnosis, specimen type and hospital ward location at the time of specimen submission were collected from hospital medical records.The medical record of the patients was accessed from October 3-7, 2022 and isolates were subculture for testing at October 10, 2022.
Healthcare workers (HCWs): Nasal swabs were collected from volunteer HCWs (doctors, nurses, laboratory personnel, and environmental cleaners) from September 12, 2021 to August 30, 2022.HCWs were recruited from target hospital units (Emergency department (ED), gynecological (GYN), medical ward (MED), pediatric ward (PEDs), surgical unit (SURG), and laboratory (LAB).HCW sample size was calculated based on a prior colonization study prevalence [15].Using single population formula, a sample size of 170 was estimated, plus an additional 20% of the sample size, for a total of 202 HCWs.Consent was obtained prior to specimen collection.The HCWs, socio-demographic information including age, sex, years of work experience, and hospital unit location was collected using a short questionnaire.Nasal swab samples were collected from the HCW.The swab was inserted approximately 2 cm into one nostril and rotated against the anterior nasal mucosa for 3 seconds.The same swab was inserted into the other nostril and rotated for 3 seconds.Upon completion, the swab was placed into the Amies transport media with charcoal.The samples were transported to SPHMMC microbiology laboratory.The samples were cultured in mannitol salt agar plate.
Hospital environmental sampling: Environmental swab samples were collected from predetermined the hospital's environment high touch or high contact surfaces and classified into three categories: Patient areas, staff areas (limited to hospital staff), and general public areas.Environmental swab specimens collected from patient areas included patient rooms (intravenous [IV] pole, bed railing, bedside table, and shelf), procedure rooms (table, medical chart, medical chart shelf), and medical supplies (oxygen, mechanical ventilator).Samples collected in staff area included nurse station (medication tray, table), documentation (file tray, medical chart), office supplies (Table, chair and computer), laboratory (chair and bench) and medical supplies and equipment (medication shelf, ultrasound).Samples were also collected from public areas: reception (shelf, hand wash sink); waiting area (chair handle and chair); corridor (elevator buttons and stair railings).Daily cleaning of the environment takes place in the morning, thus all 90 of the environmental samples were all collected after 2:00 pm.Samples were collected using two pre-moisten with sterile saline water swabs and Amies transport media were used to transport the sample to the laboratory [16].

Identification of S. aureus
Preliminary S. aureus identification was performed at SPHMMC microbiology laboratory using pre-enrichment, non-selective and selective media as previously described [16].In summary, swabs from HCWs and the environment were placed in 2 mL of tryptone soy broth with NaCl and incubated aerobically at 35˚C for 24 hours, after which 50-μL aliquots of the broth were inoculated on to mannitol salt agar plates.Colonies were selected based on colony morphology and were then sub-cultured on to tryptic soy agar (TSA).Positive catalase and coagulase reactions were considered presumptive positive for S. aureus.

Confirmation of MSSA, MRSA and antimicrobial susceptibility
Isolate confirmation and antimicrobial susceptibility testing (AST) were performed for all presumptive isolates at EPHI Clinical Bacteriology and Mycology Laboratory using BD Phoenix M50 system (BD Diagnostic Systems, Franklin Lakes, NJ, USA) version 6.8.1 PMIC/ID-111 panel following manufacturer's procedures [17].In addition, isolates were determined as either MSSA or MRSA based on the resistance to cefoxitin as determined by minimum inhibitory concentration (MIC> = 8).The isolates were stored at -80˚C in tryptic soy broth (TSB) containing 20% glycerol for further molecular testing.

Nucleic acid isolation and amplification of 16S rRNA, mecA, lukF-PV and spa genes
Genomic DNA was extracted using a commercially available QIAamp DNA Mini kit (QIA-GEN, Hilden, Germany), following the manufacturer's protocol [18].DNA extraction and polymerase chain reaction (PCR) tests were performed at EPHI.The extract was stored at -20˚C until further analysis.For PCR amplification and analysis of 16S rRNA, mecA, lukF-PV and spa genes, the QIAGEN Microbial DNA qPCR Multi-Assay Kit (QIAGEN, Hilden, Germany) was used according to the manufacturer's procedure [19].

Statistical analysis
STATA version 16.2 (Stata Corp LLC, College Station, TX, USA) was used for data analysis.Descriptive statistics were generated to summarize cefoxitin screening results and S. aureus prevalence by sampling site.Logistic regression models were fitted to calculated odds ratio and determine the association between antibiotic resistance and sample source; lukF-PV carriage and antimicrobial resistance; MRSA status and independent factors such as age, gender, departments and HCW's years of work experience.

Ethical clearance
The study protocol was approved by the EPHI Institutional Review Board (IRB, EPHI-IRB-029-2017) and SPHHMC IRB (PM23/352).Written consent was obtained from healthcare providers for colonization screening.Retrospective patient data and archived isolates were used; Anonymous analysis was conducted; authors did not have access to information that could identify individual participants, and the ethics committee waived patient consent.

Socio-demographic characteristics of study participants
Patients.Out of 73 patients with S. aureus positive culture, 44 (60.3%) were female.The female patients had a mean age of 24.1 years and a median age of 26 years.In contrast, the male patients had a mean age of 16.7 years and a median age of 7 years.For females, the age group of 26-39 made up the majority of participants, whereas for males, it was one year and younger (Table 1).

Health care workers
Out of the 202 HCWs who enrolled and consented to participate in the study, 103 (51.0%) were males.Although the age of HCW range from 21 to 49 years, the majority (65.8%) of them were in the 21-29 age group, with a mean age of 29 and a median age of 28 (Table 2).

Identification of MSSA and MRSA from patients, HCWs and environment
Overall, a total of 124 MSSA and 10 MRSA isolates were identified.Out of the 124 MSSA, 70 isolates were from patients' clinical specimen, 46 from HCW nasal colonization, and 8 from hospital environmental source (Fig 1).Of the 10 MRSA isolates, 3 were from patient's clinical specimen, 5 from HCW nasal colonization, and 2 from hospital environmental source.There was no significant difference in MRSA prevalence among different sample sources.
Of the total of 73 patient isolates, 3 (4.1%)were MRSA.The distribution of S. aureus isolation from different departments varies with higher percentage in the pediatric ward and the lowest in the outpatient department (Table 1).
Among the total of 202 HCW participants, 51 (25.2%) were colonized with S. aureus of which 5 (9.8%) were found to be MRSA.The distribution of S. aureus varied between different age groups.Higher frequencies of S. aureus and MRSA were found in the age group 21-29 years, but the difference was not statistically significant.The percentage of S. aureus and MRSA among HCWs was relatively higher in the surgery department, 11 (21.6%) and 2 (40.0%), respectively; however, this difference was not statistically significant.Similarly, there were no significant associations between isolation of S. aureus and years of work experience among HCWs (S1 Table ).Out of the 90 environmental samples, 10 (11.1%) yielded S. aureus isolates.Two of these isolates (20.0%) were MRSA.One MRSA isolate was recovered from the patient area, while the other was from the staff area.

Environment
All of the10 S. aureus strain isolated from hospital environment were resistant to both ampicillin and penicillin while 3 (30.0%)were resistant to clindamycin, erythromycin and levofloxacin.None of the environmental isolates were resistant to ceftaroline and tetracycline (Table 4).

spa, mecA, and lukF-PV gene detection
All 134 isolates tested positive for the presence of the spa genes, whereas the mecA gene was only detected in 10 (7.4%) MRSA isolates.The lukF-PV gene was detected in 92/134 (68.6%) of the S. aureus isolates, including in 62 (85.0%), 26 (51.0%) and 4 (40.0%) of the patients, healthcare worker, and environmental isolates, respectively.Four (40%) of the 10 mecA positive MRSA isolates possessed the lukF-PV gene, compared to 87/124 (70.2%) of MSSA isolates.Among the latter, 60/87 (69.0%) were isolated from patients, while 25/87 (28.7%) were isolated from health care workers and 2 (2.3%) from environmental samples.Significant associations were observed between isolates from patients and the presence of the lukF-PV gene in reference to environmental samples (Table 6).lukF-PV gene positive isolates had a 4.54 times higher probability of being SXT-resistant; while oxacillin-resistant isolates were less common among isolates that tested positive for lukF-PV gene (Table 6).However, no associations were found between lukF-PV prevalence and departmental location.

Discussion
The emergence and persistent spread of drug-resistant S. aureus have become one of the most frightening problems facing the world today.In the present study, we found that the majority of isolates were MSSA; nevertheless, these were resistant to commonly prescribed oral antibiotics such as penicillin, SXT, tetracycline, erythromycin and clindamycin resulting in limited treatment options for outpatients.In addition, over two thirds of the isolates harbored the lukF-PV gene encoding PVL, indicating increase virulence in the isolates.Association between S. aureus / MRSA infection and host attributes such as age and gender have been searched for in the present study.The most frequent age ranges for female patients were 26 to 39 years old and for male it was under 1 year of age.With the exception of four blood samples, most of the children under a year old were from outpatient and had pus or abscesses.Since the children were not hospitalized and the majority of their S. aureus infections were community associated, an outbreak in the hospital was not likely the source of the children's infection.
Male children displayed a slightly higher prevalence of S. aureus infection among other age groups.Overall, there was a slightly higher percent of female patients in our study, in contrast to other studies that have reported a higher prevalence of S. aureus infection in males [7].Majority of female patients were in the reproductive age and some of them were breast feeding (data not shown).S. aureus transmission from mother to child has been reported in different studies [20,21].Given the high infection rate among young mothers and infants found in the present study, further studies are needed in our patient population to conclude the risk of mother-to-child transmission of S. aureus.S. aureus exhibit many virulence factors that are important or necessary for its survival and ability to infect humans.One of the key components of S. aureus pathogenicity that helps it avoid human immunological reactions is staphylococcal protein A, [22].In this study, the spa gene was found in every isolate; however, in other studies, the difference in primer binding sites caused up to 3% of the isolates not to detect the gene [23].Panton-Valentine Leukocidin (PVL) is another virulence gene expressed by S. aureus and is known to be associated with severe form of infection.It is usually associated with skin and soft tissue infection and sepsis [24,25].In this study over two-third of the isolates possessed PVL gene and this is in agreement with previous study conducted in Ethiopia indicating that there is a high prevalence virulent S. aureus strains encoding pvl gene in the county [26].
S. aureus may carry the mecA gene, which confers resistance to methicillin, penicillin, and other drugs that resemble penicillin.In this study the detection of MRSA in patients was very low compared to other studies conducted in Ethiopia [12,13].Other studies utilizing molecular techniques also revealed very low frequencies of MRSA cases in Ethiopia [14,27].This may be because the majority of the study conducted used Kirby-Bauer disc diffusion method by cefoxitin screening or oxacillin test (which needs E-test for confirmation and most of the laboratories do not have E tests).Similar condition was reported in other East Africa country [28].
Interestingly, the age range of the majority of HCWs was between 21 and 29 years of age, indicating a young workforce.This is consistent with the report that showed the majority of healthcare professionals in Ethiopia were under 35 years old [29].This could be the cause of the lack of significant association between MRSA carriage and age group.The percentage of S. aureus identification was comparable to other studies in Ethiopia [30,31].The HCWs' department and year of experience had no significant association with the presence of MRSA.In contrast, other studies investigating the nasal carriage of S. aureus and MRSA, reported significant associations with one or more of the aforementioned factors [31,32].Similarly, there was no statistically significant association between the hospital ward and the sample collection area among environmental isolates of MRSA.
In environmental samples, the frequency of MRSA was slightly higher.In addition, fluoroquinolones have shown significantly high level of resistance in environmental samples compared to other samples sources.This may be because the bacteria in a hospital environment are more likely to develop antibiotic resistance due to high use [33,34].A study conducted elsewhere reported high MRSA contamination in hospital surfaces during no outbreak periods [35].A study conducted in another hospital in Addis Ababa reported a comparable MRSA colonization rate of 9.8% among health care workers [31].
In this investigation, all S. aureus isolates were susceptible to antibiotics such as daptomycin, linezolid, nitrofurantoin, and vancomycin.These findings were in line with those of other East African studies [14,36,37].For instance, linezolid is only authorized for use in certain centers and is only designated for the treatment of multidrug-resistant tuberculosis [38].Other antibiotics, such as daptomycin, are not included in the national drug list [38].
Since every isolate was responsive to the previously listed antibiotics, daptomycin and vancomycin are prescribed for serious infections such as bacteremia/ osteomyelitis caused by MRSA or for those with penicillin allergy who have MSSA and are unable to take oxacillin.It ensures that patients with severe invasive S. aureus infections may still have recourse to potent antibiotics.By contrast, a substantial percentage of regularly used antibiotics, including oral medications such as penicillin, SXT, and tetracycline, demonstrated high levels of resistance.Studies carried out elsewhere in Ethiopia have revealed a similar resistance profile [11,14].This is likely connected to self-medication and overuse of antibiotics in community settings [37,39].There are also reports indicating that many antibiotics are prescribed empirically and unnecessarily [40,41].
An isolate from HCW sample displayed resistance to oxacillin but was susceptible to cefoxitin.Cefoxitin is more reliable indicator of mecA gene carriage, and molecular analysis confirmed that this isolate did not harbor the mecA gene.This finding highlights that resistance to oxacillin is mediated by resistance mechanisms other than mecA [42].
Despite the fact that ceftaroline has been shown to have increased activity against MRSA in many countries globally [14,43,44], it is concerning that we have identified ceftaroline resistant isolates.Especially because this antibiotic is not included in Ethiopia's list of essential medications and is not available at SPHMMC [38].Ceftaroline may be available in some hospitals in Addis Ababa and this resistance could be derived from selection pressure.It is also alarming that of the two MRSA isolates with intermediate resistance to ceftaroline, one originated from a patient and the other from a healthcare provider.
The virulence gene lukF-PV was present in around two-thirds of the isolates, including 40% of the MRSA isolates.When compared isolates from other sources, patient sample isolates displayed greater levels of resistance to SXT which demonstrated statistically significant associations.The detection of the virulence gene (lukF-PV) was relatively higher across all sample sources, with patient samples displaying the most significant statistical correlation.This may be linked to the possibility that isolates that produce infection are more virulent as compared to strains identified in environment.A study from East Africa suggested that the pvl toxin may be increasing in prevalence among clinical isolates [45].
Whereas a previous study in Ethiopia did not find the lukF-PV gene in MRSA isolates, this study observed the lukF-PV gene in mecA positive isolates [46].Similar reports from other East African countries suggest that MRSA strains harboring PVL represent significant pathogenic strains in patients with skin and soft tissue infections [47].

Conclusion
Although, some of the antibiotics such as vancomycin, linezolid and daptomycin were complete susceptible to S. aureus, there is higher resistance against common antibiotics such as penicillin, trimethoprim sulfamethoxazole and erythromycin and clindamycin.Ceftaroline resistance is concerning as well, and since testing for resistance is not regularly done, additional study is required to determine the true prevalence of the resistance.The prevalence of MRSA found in patients, healthcare workers, and the environment was comparatively low.The detection of MSSA or MRSA was shown to have no association with sample source, sample collection department, gender and age.Moreover, patient isolates had significant association with high prevalence of lukF-PV gene.These findings demonstrate the emergence of antimicrobial resistance to new drugs and the prevalence of virulent S. aureus strains in the study site.Further studies to identify potential transmission links and improved infection prevention and control and environmental cleaning are needed to prevent spread.In addition, we recommend enforcing proper use and regulation of antibiotics in Ethiopia.

Table 5
below shows the association between antimicrobial resistance and sample source (patients vs. healthcare workers in reference to environmental sample) for several different antibiotics.The table includes odds ratios (OR) and p-values for each antibiotic.Significantly high level of resistance was observed in levofloxacin and ciprofloxacin in environmental samples compared to patient's sample.While the odds of S. aureus to be resistant to SXT among patients are 6.81times more, that is statically significant.